Immobilized molecular synthesis of systematically substituted compounds

ABSTRACT

A synthetic strategy for the creation of large scale chemical diversity is claimed. Solid-phase chemistry, photolabile protecting groups, and photolithography are used to achieve light-directed spatially-addressable parallel chemical synthesis of an array of polymers that are based on a target polymer. The array includes systematically substituted versions of the target polymer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.796,727, filed Nov. 22, 1991 now U.S. Pat. No. 5,242,974. Thisapplication is also a continuation-in-part of U.S. application Ser. No.805,727, filed Dec. 6, 1991 now U.S. Pat. No. 5,424,186, and priority isclaimed from these applications. All of the above identifiedapplications are incorporated herein by reference for all purposes.

U.S. application Ser. No. 805,727 is a continuation-in-part of U.S.application Ser. No. 492,462 (now U.S. Pat. No. 5,143,854), filed Mar.7, 1990, which is a continuation-in-part of U.S. application Ser. No.362,901, filed Jun. 7, 1989 (abandoned). No priority is claimed fromthese applications.

This application is also related to the following U.S. application Ser.Nos. 626,730 and 624,114, both filed Dec. 6, 1990; and U.S. applicationSer. Nos. 796,243 and 796,947, both filed on Nov. 22, 1991 now U.S. Pat.Nos. 5,384,261 and 5,324,633, respectively. No priority is claimed fromthese applications and patents. Each of these applications isincorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

The present invention relates to the field of molecular synthesis. Morespecifically, the invention provides systems and methods for directedsynthesis of diverse molecular sequences on substrates.

Methods for preparing different polymers are well known. For example,the "Merrifield" method, described in Atherton et al., "Solid PhasePeptide Synthesis," IRL Press, 1989, which is incorporated herein byreference for all purposes, has been used to synthesize peptides on asolid support. In the Merrifield method, an amino acid is covalentlybonded to a support made of an insoluble polymer or other material.Another amino acid with an alpha protecting group is reacted with thecovalently bonded amino acid to form a dipeptide. After washing, theprotecting group is removed and a third amino acid with an alphaprotecting group is added to the dipeptide. This process is continueduntil a peptide of a desired length and sequence is obtained.

Other techniques have also been described. These methods include thesynthesis of peptides on 96 plastic pins which fit the format ofstandard microtiter plates. Advanced techniques for synthesizing largenumbers of molecules in an efficient manner have also been disclosed.Most notably, U.S. Pat. No. 5,143,854 (Pirrung et al.) and PCTApplication No. 92/10092 disclose improved methods of molecularsynthesis using light directed techniques. According to these methods,light is directed to selected regions of a substrate to removeprotecting groups from the selected regions of the substrate.Thereafter, selected molecules are coupled to the substrate, followed byadditional irradiation and coupling steps.

SUMMARY OF THE INVENTION

Methods, devices, and compositions for synthesis and use of diversemolecular sequences on a substrate are disclosed, as well asapplications thereof.

A preferred embodiment of the invention provides for the synthesis of anarray of polymers in which individual monomers in a lead polymer aresystematically substituted with monomers from one or more basis sets ofmonomers. The method requires a limited number of masks and a limitednumber of processing steps. According to one specific aspect of theinvention, a series of masking steps are conducted to first place thefirst monomer in the lead sequence on a substrate at a plurality ofsynthesis sites. The second monomer in the lead sequence is then addedto the first monomer at a portion of the synthesis sites, whiledifferent monomers from a basis set are placed at discrete othersynthesis sites. The process is repeated to produce all or a significantnumber of the mono substituted polymers based on the lead polymer usinga given basis set of monomers. According to a preferred aspect of theinvention, the technique uses light directed techniques, such as thosedescribed in Pirrung et al., U.S. Pat. No. 5,143,854.

Another aspect of the invention provides for efficient synthesis andscreening of cyclic molecules. According to a preferred aspect of theinvention, cyclic polymers are synthesized in an array in which thepolymers are coupled to the substrate at different positions on thecyclic polymer ring. Therefore, a particular polymer may be presented invarious "rotated" forms on the substrate for later screening. Again, thecyclic polymers are formed according to most preferred embodiments withthe techniques of Pirrung et al.

The resulting substrates will have a variety of uses including, forexample, screening polymers for biological activity. To screen forbiological activity, the substrate is exposed to one or more receptorssuch as an antibody, oligonucleotide, whole cells, receptors onvesicles, lipids, or any one of a variety of other receptors. Thereceptors are preferably labeled with, for example, a fluorescentmarker, a radioactive marker, or a labeled antibody reactive with thereceptor. The location of the marker on the substrate is detected with,for example, photon detection or auto-radiographic techniques. Throughknowledge of the sequence of the material at the location where bindingis detected, it is possible to quickly determine which polymer(s) arecomplementary with the receptor. The technique can be used to screenlarge numbers of peptides or other polymers quickly and economically.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C illustrate a systematic substitution masking strategy;

FIG. 2 illustrates additional aspects of a systemmatic substitutionmasking strategy;

FIGS. 3, 4, and 5 illustrate rotated cyclic polymer groups;

FIGS. 6A to 6E illustrate formation of rotated cyclic polymers;

FIGS. 7A to 7C illustrate formation of rotated and substituted cyclicpolymers;

FIG. 8 illustrates the array of cyclic polymers resulting from thesynthesis;

FIG. 9 illustrates masks used in the synthesis of cyclic polymer arrays;

FIGS. 10A and 10B illustrate coupling of a tether in two orientations;

FIGS. 11A and 11B illustrate masks used in another embodiment;

FIGS. 12A and 12B show a tripeptide used in a fluorescenceenergy-transfer substrate assay and that substrate after cleavage;

FIGS. 13A to 13H illustrate donor/quencher pairs;

FIG. 14 illustrates sequence versus normalized fluorescence intensityfor the ten possible single deletion peptides binding to the D32.39antibody. A blank space represents a deleted amino acid relative to thefull length kernel sequence (FLRRQFKVVT) (SEQ ID NO: 1) shown on thebottom. Error bars represent the standard deviation of the averagedsignals from four replicates. All peptides are acetylated on the aminoterminus and are linked to the surface via an amide bond to the carboxylterminus; and

FIG. 15 illustrates sequence versus normalized fluorescence intensityfor the terminally truncated peptides. The full length kernal sequence(FLRQFKVVT) (SEQ. ID NO: 2) is shown in the center of the graph. Errorbars represent the standard deviation of the averaged signals from aminumum of four replicates. All peptides are acetylated on the aminoterminus and are linked to the surface via an amide bond to the carboxylterminus.

DESCRIPTION OF THE PREFERRED EMBODIMENTS CONTENTS I. Definitions II.Synthesis

A. Systematic Substitution

B. Cyclic Polymer Mapping

III. Data Collection

A. CCD Data Collection System

B. Trapping Low Affinity Interactions

C. Fluorescence Energy-Transfer Substrate Assays

IV. Examples

A. Example

B. Example

V. Conclusion I. Definitions

Certain terms used herein are intended to have the following generaldefinitions:

1. Complementary: This term refers to the topological compatibility ormatching together of interacting surfaces of a ligand molecule and itsreceptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

2. Epitope: An epitope is that portion of an antigen molecule which isdelineated by the area of interaction with the subclass of receptorsknown as antibodies.

3. Ligand: A ligand is a molecule that is recognized by a particularreceptor. Examples of ligands that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones, hormonereceptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g.,opiates, steroids, etc.), lectins, sugars, oligonucleotides (such as inhybridization studies), nucleic acids, oligosaccharides, proteins,benzodiazapines, prostaglandins, beta-turn mimetics, and monoclonalantibodies.

4. Monomer: A monomer is a member of the set of smaller molecules whichcan be joined together to form a larger molecule. The set of monomersincludes but is not restricted to, for example, the set of commonL-amino acids, the set of D-amino acids, the set of natural or syntheticamino acids, the set of nucleotides and the set of pentoses and hexoses.As used herein, monomer refers to any member of a basis set forsynthesis of a larger molecule. A selected set of monomers forms a basisset of monomers. For example, dimers of the 20 naturally occurringL-amino acids form a basis set of 400 monomers for synthesis ofpolypeptides. Different basis sets of monomers may be used in any of thesuccessive steps in the synthesis of a polymer. Furthermore, each of thesets may include protected members which are modified after synthesis.

5. Peptide: A peptide is a polymer in which the monomers are natural orunnatural amino acids and which are joined together through amide bonds,alternatively referred to as a polypeptide. In the context of thisspecification, it should be appreciated that the amino acids may be, forexample, the L-optical isomer or the D-optical isomer. Specificimplementations of the present invention will result in the formation ofpeptides with two or more amino acid monomers, often 4 or more aminoacids, often 5 or more amino acids, often 10 or more amino acids, often15 or more amino acids, and often 20 or more amino acids. Standardabbreviations for amino acids are used (e.g., P for proline). Theseabbreviations are included in Stryer, Biochemistry, Third Ed., 1988,which is incorporated herein by reference for all purposes.

6. Radiation: Radiation is energy which may be selectively applied,including energy having a wavelength of between 10⁻¹⁴ and 10⁴ metersincluding, for example, electron beam radiation, gamma radiation, x-rayradiation, light such as ultra-violet light, visible light, and infraredlight, microwave radiation, and radio waves. "Irradiation" refers to theapplication of radiation to a surface.

7. Receptor: A receptor is a molecule that has an affinity for a givenligand. Receptors may be naturally-occurring or synthetic molecules.Also, they can be employed in their unaltered state, in derivativeforms, or as aggregates with other species. Receptors may be attached,covalently or noncovalently, to a binding member, either directly or viaa specific binding substance. Examples of receptors which can beemployed by this invention include, but are not restricted to,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses,cells, or other materials), drugs, oligonucleotides, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles. Receptors are sometimesreferred to in the art as anti-ligands. As the term receptors is usedherein, no difference in meaning is intended. A "Ligand Receptor Pair"is formed when two molecules have combined through molecular recognitionto form a complex.

Other examples of receptors which can be investigated by this inventioninclude but are not restricted to microorganism receptors, enzymes,catalytic polypeptides, hormone receptors, and opiate receptors.

8. Substrate: A substrate is a material having a rigid or semi-rigidsurface, generally insoluble in a solvent of interest such as water,porous and/or non-porous. In many embodiments, at least one surface ofthe substrate will be substantially flat, although in some embodimentsit may be desirable to physically separate synthesis regions fordifferent polymers with, for example, wells, raised regions, etchedtrenches, or the like. According to other embodiments, small beads maybe provided on the surface which may be released upon completion of thesynthesis.

9. Protecting group: A protecting group is a material which ischemically bound to a monomer unit or polymer and which may be removedupon selective exposure to an activator such as electromagneticradiation or light, especially ultraviolet and visible light. Examplesof protecting groups with utility herein include those comprisingortho-nitro benzyl derivatives, nitropiperonyl, pyrenylmethoxy-carbonyl,nitroveratryl, nitrobenzyl, dimethyl dimethoxybenzyl,5-bromo-7-nitroindolinyl, o-hydroxy-α-methyl cinnamoyl, and2-oxymethylene anthraquinone.

10. Predefined Region: A predefined region is a localized area on asurface which is, was, or is intended to be activated for formation of amolecule using the techniques described herein. The predefined regionmay have any convenient shape, e.g., circular, rectangular, elliptical,wedge-shaped, etc. For the sake of brevity herein, "predefined regions"are sometimes referred to simply as "regions." A predefined region maybe illuminated in a specified step, along with other regions of asubstrate.

11. Substantially Pure: A molecule is considered to be "substantiallypure" within a predefined region of a substrate when it exhibitscharacteristics that distinguish it from other predefined regions.Typically, purity will be measured in terms of biological activity orfunction as a result of uniform sequence. Such characteristics willtypically be measured by way of binding with a selected ligand orreceptor. Preferably the region is sufficiently pure such that thepredominant species in the predefined region is the desired sequence.According to preferred aspects of the invention, the molecules formedare 5% pure, more preferably more than 10% pure, preferably more than20% pure, more preferably more than 80% pure, more preferably more than90% pure, more preferably more than 95% pure, where purity for thispurpose refers to the ratio of the number of ligand molecules formed ina predefined region having a desired sequence to the total number ofmolecules formed in the predefined region.

12. Activator: A activator is a material or energy source adapted torender a group active and which is directed from a source to at least apredefined location on a substrate, such as radiation. A primaryillustration of an activator is light, such as visible, ultraviolet, orinfrared light. Other examples of activators include ion beams, electricfields, magnetic fields, electron beams, x-ray, and the like.

13. Combinatorial Synthesis Strategy: A combinatorial synthesis strategyis an ordered strategy for parallel synthesis of diverse polymersequences by sequential addition of reagents which may be represented bya reactant matrix and a switch matrix, the product of which is a productmatrix. A reactant matrix is a l column by m row matrix of the buildingblocks to be added. The switch matrix is all or a subset of the binarynumbers, preferably ordered, between l and m arranged in columns. A"binary strategy" is one in which at least two successive stepsilluminate a portion, often half, of a region of interest on thesubstrate. In a binary synthesis strategy, all possible compounds whichcan be formed from an ordered set of reactants are formed. In mostpreferred embodiments, binary synthesis refers to a synthesis strategywhich also factors a previous addition step. For example, a strategy inwhich a switch matrix for a masking strategy halves regions that werepreviously illuminated, illuminating about half of the previouslyilluminated region and protecting the remaining half (while alsoprotecting about half of previously protected regions and illuminatingabout half of previously protected regions). It will be recognized thatbinary rounds may be interspersed with non-binary rounds and that only aportion of a substrate may be subjected to a binary scheme. Acombinatorial "masking" strategy is a synthesis which uses light orother spatially selective deprotecting or activating agents to removeprotecting groups from materials for addition of other materials such asamino acids.

14. Linker: A linker is a molecule or group of molecules attached to asubstrate, and spacing a synthesized polymer from the substrate forexposure/binding to a receptor.

15. Systematically Substituted: A position in a target molecule has beensystematically substituted when the molecule is formed at a plurality ofsynthesis sites, with the molecule having a different member of a basisset of monomers at the selected position of the molecule within each ofthe synthesis sites on the substrate.

16. Abbreviations: The following frequently used abbreviations areintended to have the following meanings:

BOC: t-butyloxycarbonyl.

BOP: benzotriazol-1-yloxytris-(dimethylamino) phosphoniumhexafluorophosphate.

DCC: dicyclohexylcarbodiimide.

DCM: dichloromethane; methylene chloride.

DDZ: dimethoxydimethylbenzyloxy.

DIEA: N,N-diisopropylethylamine.

DMAP: 4-dimethylaminopyridine.

DMF: dimethyl formamide.

DMT: dimethoxytrityl.

FMOC: fluorenylmethyloxycarbonyl.

HBTU: 2-(1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluroniumhexafluorophosphate.

HOBT: 1-hydroxybenzotriazole.

NBOC: 2-nitrobenzyloxycarbonyl.

NMP: N-methylpyrrolidone.

NPOC: 6-nitropiperonyloxycarbonyl.

NV: 6-nitroveratryl.

NVOC: 6-nitroveratryloxycarbonyl.

PG: protecting group.

TFA: trifluoracetic acid.

THF: tetrahydrofuran.

II. Synthesis

The present invention provides synthetic strategies and devices for thecreation of large scale chemical diversity. Solid-phase chemistry,photolabile protecting groups, and photolithography are brought togetherto achieve light-directed spatially-addressable parallel chemicalsynthesis in preferred embodiments.

The invention is described herein for purposes of illustration primarilywith regard to the preparation of peptides and nucleotides but couldreadily be applied in the preparation of other molecules. Such moleculesinclude, for example, both linear and cyclic polymers of nucleic acids,polysaccharides, phospholipids, and peptides having either α-, β-, orω-amino acids, heteropolymers in which a known drug is covalently boundto any of the above, polyurethanes, polyesters, polycarbonates,polyureas, n-alkylureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, carbamates, sulfones, sulfoxides,polyacetates, or other polymers which will be apparent upon review ofthis disclosure. It will be recognized further that peptideillustrations herein are primarily with reference to C- to N-terminalsynthesis, but the invention could readily be applied to N- toC-terminal synthesis without departing from the scope of the invention.Methods for forming cyclic and reversed polarity peptides and otherpolymers are described in copending application Ser. No. 796,727, filedNov. 22, 1991, and previously incorporated herein by reference. Othermolecules that are not conventionally viewed as polymers but which areformed from a basis set of monomers or building blocks may also beformed according to the invention herein.

The prepared substrate may, for example, be used in screening a varietyof polymers as ligands for binding with a receptor, although it will beapparent that the invention could be used for the synthesis of areceptor for binding with a ligand. The substrate disclosed herein willhave a wide variety of uses. Merely by way of example, the inventionherein can be used in determining peptide and nucleic acid sequencesthat bind to proteins, finding sequence-specific binding drugs,identifying epitopes recognized by antibodies, and evaluating a varietyof drugs for clinical and diagnostic applications, as well ascombinations of the above.

The invention preferably provides for the use of a substrate "S" with asurface. Linker molecules "L" are optionally provided on a surface ofthe substrate. The purpose of the linker molecules, in some embodiments,is to facilitate receptor recognition of the synthesized polymers.

Optionally, the linker molecules are chemically protected for storage orsynthesis purposes. A chemical protecting group such as t-BOC(t-butyloxycarbonyl) is used in some embodiments. Such chemicalprotecting groups would be chemically removed upon exposure to, forexample, acidic solution and could serve, inter alia to protect thesurface during storage and be removed prior to polymer preparation.

When a polymer sequence to be synthesized is, for example, apolypeptide, amino groups at the ends of linkers attached to a glasssubstrate are derivatized with, for example, nitroveratryloxycarbonyl(NVOC), a photoremovable protecting group. The linker molecules may be,for example, aryl acetylene, ethylene glycol oligomers containing from2-10 monomers, diamines, diacids, amino acids, or combinations thereof.

According to one aspect of the invention, on the substrate or a distalend of the linker molecules, a functional group with a protecting groupP₀ is provided. The protecting group P₀ may be removed upon exposure toan activator such as a chemical reagent, radiation, electric fields,electric currents, or other activators to expose the functional group.In a preferred embodiment, the radiation is ultraviolet (UV), infrared(IR), or visible light, or a basic or acidic reagent. In still furtheralternative embodiments, ion beams, electron beams, or the like may beused for deprotection.

Photodeprotection is effected by illumination of the substrate through,for example, a mask wherein the pattern produces illuminated regionswith dimensions of, for example, less than 1 cm², 10⁻¹ cm², 10⁻² cm²,10⁻³ cm², 10⁻⁴ cm², 10⁻⁵ cm², 10⁻⁶ cm², 10⁻⁷ cm², 10⁻⁸ cm², or 10⁻¹⁰cm². In a preferred embodiment, the regions are between about 10×10 μmand 500×500 μm. According to some embodiments, the masks are arranged toproduce a checkerboard array of polymers, although any one of a varietyof geometric configurations may be utilized.

Concurrently with or after exposure of a known region of the substrateto light or another activator, the surface is contacted with a firstmonomer unit M₁ which reacts with the functional group that has beenexposed by the deprotection step. The first monomer includes aprotecting group P₁. P₁ may or may not be the same as P₀.

Accordingly, after a first cycle, first regions of the surface comprisethe sequence:

    S-L-M.sub.1 -P.sub.1

while remaining regions of the surface comprise the sequence:

    S-L-P.sub.0.

Thereafter, one or more second regions of the surface (which may includeall or part of the first region, as well as other regions) are exposedto light and contacted with a second monomer M₂ (which may or may not bethe same as M₁) having a protecting group P₂. P₂ may or max not be thesame as P₀ and P₁. After this second cycle, different regions of thesubstrate may comprise one or more of the following sequences:

    S- L-M.sub.1 -M.sub.2 -P.sub.2

    S-L-M.sub.2 -P.sub.2

    S-L-M.sub.1 -P.sub.1 and/or

    S-L-P.sub.0.

The above process is repeated until the substrate includes desiredpolymers of desired lengths. By controlling the locations of thesubstrate exposed to light and the reagents exposed to the substratefollowing exposure, one knows the location of each sequence.

According to some embodiments of the invention, multiple protectinggroups are utilized. For example, when light-labile protecting groupsare utilized to protect the growing polymer chain, it will be desirablein some embodiments to provide different protecting groups on at leastselected side groups of the various monomers. For example, acid or baselabile protecting groups may be particularly desirable when light labileprotecting groups are used on the growing polymer chain. As a specificexample, in the case of amino acids, the sulfhydryl groups of cysteineside chains can form disulfide bonds with one another. Accordingly, itwill sometimes be desirable to protect such side groups with an acid orbase labile protecting group, or a protecting group that is removed witha wavelength of light different from that which removes the protectinggroup on the growing polymer chain. Then, one can selectively couplethese side chains by removing the appropriate protecting groups.

Thereafter, the protecting groups are removed from some or all of thesubstrate and the sequences are, optionally, capped with a capping unitC. The process results in a substrate having a surface with a pluralityof polymers of the following general formula:

    S-[L]-(M.sub.i)-(M.sub.j)-(M.sub.k) . . . (M.sub.x)-[C]

where square brackets indicate optional groups, and M_(i) . . . M_(x)indicates any sequence of monomers. The number of monomers could cover awide variety of values, but in a preferred embodiment they will rangefrom 2 to 100.

In some embodiments, a plurality of locations on the substrate polymerscontain a common monomer subsequence. For example, it may be desired tosynthesize a sequence S-M₁ -M₂ -M₃ at first locations and a sequenceS-M₄ -M₂ -M₃ at second locations. The process would commence withirradiation of the first locations followed by contacting with M₁ -P,resulting in the sequence S-M₁ -P at the first location. The secondlocations would then be irradiated and contacted with M₄ -P, resultingin the sequence S-M₄ -P at the second locations. Thereafter both thefirst and second locations would be irradiated and contacted withmonomers M₂ and M₃ (or with the dimer M₂ -M₃, resulting in the sequenceS-M₁ -M₂ -M₃ at the first locations and S-M₄ -M₂ -M₃ at the secondlocations. Of course, common subsequences of any length could beutilized including those in a range of 2 or more monomers, such as 2 to10 monomers, 2 to 20 monomers, or 2 to 100 monomers.

The polymers prepared on a substrate according to the above methods willhave a variety of uses including, for example, screening for biologicalactivity, i.e., such as ability to bind to a receptor. In such screeningactivities, the substrate containing the sequences is exposed to anunlabeled or labeled receptor such as an antibody, a receptor on a cell,a phospholipid vesicle, or any one of a variety of other receptors. Inone preferred embodiment, the polymers are exposed to a first, unlabeledor labeled receptor of interest and thereafter exposed to a labeledreceptor-specific recognition element, which is, for example, anantibody. This process can provide signal amplification in the detectionstage.

The receptor molecules may or may not bind with one or more polymers onthe substrate. The presence (or lack thereof) of the labeled receptorand, therefore, the presence of a sequence which binds with the receptoris detected in a preferred embodiment through the use ofautoradiography, detection of fluorescence with a charge-coupled device,fluorescence microscopy, or the like. The sequence of the polymer at thelocations where the receptor binding is detected may be used todetermine all or part of a sequence which is complementary to thereceptor.

Use of the invention herein is illustrated primarily with reference toscreening for binding to a complementary receptor. The invention will,however, find many other uses. For example, the invention may be used ininformation storage (e.g., on optical disks), production of molecularelectronic devices, production of stationary phases in separationsciences, production of dyes and brightening agents, photography, and inimmobilization of cells, proteins, lectins, nucleic acids,polysaccharides, and the like in patterns on a surface via molecularrecognition of specific polymer sequences. By synthesizing the samecompound in adjacent, progressively differing concentrations, one canestablish a gradient to control chemotaxis or to develop diagnostic"dipsticks," which, for example, titrate an antibody against anincreasing amount of antigen. By synthesizing several catalyst moleculesin close proximity, one can achieve more efficient multistep conversionsby "coordinate immobilization." Coordinate immobilization also may beused for electron transfer systems, as well as to provide bothstructural integrity and other desirable properties to materials, suchas lubrication, wetting, etc.

According to alternative embodiments, molecular biodistribution orpharmacokinetic properties may be examined. For example, to assessresistance to intestinal or serum proteases, polymers may be capped witha fluorescent tag and exposed to biological fluids of interest.

A high degree of miniaturization is possible, because the density ofcompounds on the surface is determined largely with regard to spatialaddressability of the activator, in one case the diffraction of light.Each compound is physically accessible and its position is preciselyknown. Hence, the array is spatially-addressable, and its interactionswith other molecules can be assessed.

According to one aspect of the invention, reactions take place in anappropriate reaction chamber that includes isolated fluid flow paths forheating or cooling liquids that are used to maintain the reactionchamber temperature at a desired level. In still further embodiments thereaction chamber is placed on a rotating "centrifuge" to reduce thevolume of reactants needed for the various coupling/deprotection stepsdisclosed herein. In a centrifuge flow cell, the substrate is placed inthe centrifuge such that, for example, when a monomer solution passesover the surface of the substrate a relatively thin film of the materialis formed on the substrate due to the higher gravitational forces actingon the substrate. Accordingly, the volume of various reagents needed inthe synthesis will be substantially reduced.

A. Systematic Substitution

According to one preferred embodiment of the invention, a "lead"sequence is identified using either the light directed techniquesdescribed herein, or more conventional methods such as those describedin Geysen, J. Imm. Methods (1987) 102:259-274, incorporated herein byreference for all purposes, or through other knowledge of the structureof the receptor in question, such as through computer modelinginformation. As used herein, a "lead" or "kernel" sequence is a moleculehaving a monomer sequence which has been shown to exhibit at leastlimited binding affinity with a receptor or class of receptors.

Thereafter, a series of molecules related to the lead sequence aregenerated by systematic substitution, deletion, addition, or acombination of these processes at one or more positions of the molecule.A sequence with a binding affinity higher than the lead sequence can be(or may be) identified through evaluation of the molecules produced bythese processes.

One aspect of the invention herein provides for improved methods forforming molecules with systematically substituted monomers or groups ofmonomers using a limited number of synthesis steps. Like the otherembodiments of the invention described herein, this aspect of theinvention has applicability not only to the evaluation of peptides, butalso other molecules, such as oligonucleotides and polysaccharides.Light-directed techniques are utilized in preferred embodiments becauseof the significant savings in time, labor, and the like.

According to one aspect of the invention, a lead polymer sequence isidentified using conventional techniques or the more sophisticated lightdirected techniques described herein. The lead sequence is generallyrepresented herein by:

    ABCDEFG

where the various letters refer to amino acids or other monomers intheir respective positions in the lead sequence. Although a polymer withseven monomers is used herein for the purpose of illustration, a largeror smaller number of monomers will typically be found in the leadpolymer in most embodiments of the invention.

Using a selected basis set of monomers, such as twenty amino acids orfour nucleotides, one generates the following series of systematicallysubstituted polymers. The sequence of the molecules generated isdetermined with reference to the columns of the map. In other words, the"map" below can be viewed as a cross-section of the substrate:

    ______________________________________                                        G        G     G         G   G       G   X                                    F        F     F         F   F       X   F                                    E        E     E         E   X       E   E                                    D        D     D         X   D       D   D                                    C        C     X         C   C       C   C                                    B        X     B         B   B       B   B                                    X        A     A         A   A       A   A                                    ______________________________________                                    

where X represents the monomers in a basis set of monomers such astwenty amino acids. For example, the twenty polymers XBCDEFG aregenerated within 20 individual synthesis sites on the substrate. In thecase of 7-monomer lead peptides, the total number of peptides generatedwith all twenty monomers in the basis set is 140, i.e., 7*20, with 134unique sequences being made and 7 occurences of the lead sequence.

One of the least efficient ways to form this array of polymers would bevia conventional synthesis techniques, which would require about 938coupling steps (134 peptides * 7 residues each). At the other extreme,each of these sequences could be made in 7 steps, but the sequenceswould be physically mixed, requiring separation after screening.

By contrast, this aspect of the invention provides for efficientsynthesis of substituted polymers. FIG. 1A-1C illustrates the maskingstrategy for the 7-monomer lead polymer. The particular masking strategyillustrated in FIG. 1A-1C utilizes rectangular masks, but it will beapparent that other shapes of masks may also be used without departingfrom the scope of the invention herein. Masking techniques whereinregions of a substrate are selectively activated by light are describedherein by way of a preferred embodiment. The inventions herein are notso limited, however, and other activation techniques may be utilized.For example, mechanical techniques of activation/coupling such asdescribed in copending application Ser. No. 07/796,243 are used in someembodiments.

As shown in FIG. 1A, the process begins by exposing substantially all ofa predefined region of the substrate to light with a mask 291, exposingapproximately 6/7 of the region of interest 289. This step is followedby exposure of the substrate to monomer A. Thereafter, a mask 292 isused to expose approximately 5/7 of the region of interest, followed bycoupling of B. It will be recognized that mask 292 may in fact be thesame mask as 291 but translated across the substrate. Accordingly,regions indicated by dashed line 290, may also be exposed to light inthis step, as well as in later steps. Thereafter, subsequent maskingsteps expose 4/7, 3/7, 2/7, and 1/7 of the area of interest on thesubstrate, each mask being used to couple a different monomer (C, D, E,F) to the substrate. The resulting substrate is schematicallyillustrated in the bottom portion of FIG. 1A along with the resultingpolymer sequences thereon. Again, the composition of the sequences onthe substrate is given by the vertical column such as "ABCDEF." As seen,1- to 6 -membered truncated portions of the target ABCDEFG are formed.

FIG. 1B illustrates the next series of masking steps. As shown in FIG.1B, the same mask 293 is used for each masking step, but the mask istranslated with respect to the substrate in each step. In each step, themask illuminates a portion of each of the "stripes" of polymers formedin FIG. 1A, and in each step a different one of the monomers in a basisset is coupled to the substrate. The mask is then translated downwardsfor irradiation of the substrate and coupling of the next monomer. Inthe first step, the mask exposes the top 1/20 of each "stripe" ofpolymers shown in FIG. 1A, and monomer X₁ is coupled to this region. Inthe second step, the mask is translated downwards and X₂ is coupled,etc. The resulting substrate is shown in the bottom portion of FIG. 1B.An additional stripe 295 is formed adjacent the region addressed in FIG.1A, this region containing a series of subregions, each containing oneof the 20 monomers in this particular basis set.

Accordingly, after the steps shown in FIG. 1B, the substrate containsthe following polymer sequences on the surface thereof (columns againindicating the sequences formed on the substrate):

    ______________________________________                                                                                 X                                                                         X   F                                                                 X       E   E                                                             X   D       D   D                                                   X         C   C       C   C                                             X     B         B   B       B   B                                    X        A     A         A   A       A   A                                    ______________________________________                                    

where X indicates that an individual region contains one of each of themonomers in the basis set. Accordingly, for example, each of the 20dimers AX are generated when the basis set is the 20 natural L-aminoacids typically found in proteins. The 20 trimers of ABX, 4-mers ofABCX, etc., are also formed at predefined regions on the substrate.

Thereafter, as shown in FIG. 1C, the process continues, optionally usingthe same mask(s) used in FIG. 1A. The masks differ only in that theyhave been translated with respect to the substrate. In step 27, monomerB is added to the substrate using the mask that illuminates only theright 1/7 of the region of the substrate of interest. In step 28, theright 2/7 of the substrate is exposed and monomer C is coupled, etc. Asshown in the bottom portion of FIG. 1C, the process results in thegeneration of all possible polymers based on the polymer ABCDEFG,wherein each monomer position is systematically substituted with allpossible monomers from a basis set.

A number of variations of the above technique will be useful in someapplications. For example, in some embodiments the process is variedslightly to form disubstitutions of a lead polymer in which thesubstitutions are in adjacent locations in the polymer. Such arrays areformed by one of a variety of techniques, but one simple techniqueprovides for each of the masks illustrated in steps 7-26 of FIG. 1 tooverlap the previous mask by some fraction, e.g., 1/3, of its height.According to such embodiments, the following array of polymers would begenerated, in addition to the previous array of 134:

    ______________________________________                                        G        G     G         G   G       G   X                                    F        F     F         F   F       X   X                                    E        E     E         E   X       X   F                                    D        D     D         X   X       E   E                                    B        X     X         C   C       C   C                                    X        X     B         B   B       B   B                                    X        A     A         A   A       A   A                                    ______________________________________                                    

The scheme may be expanded to produce tri-substituted,tetra-substituted, etc. molecules. Accordingly, the present inventionprovides a method of forming all molecules in which at least onelocation in the polymer is systematically substituted with all possiblemonomers from a basis set.

According to a preferred aspect of the invention, masks are formed andreused to minimize the number of masks used in the process. FIG. 2illustrates how masks may be designed in this manner. For simplicity, a5-monomer synthesis is illustrated. The masks are illustrated from"above" in FIG. 2, with the cross hatching indicating light-transmissiveregions. The resulting substrate is shown in the bottom portion of FIG.2, with the region of primary interest for mono-substitutions indicatedby the arrows.

FIG. 2 illustrates how to generate and systematically substitute all ofthe 5-mers contained in a 6-mer lead. A limited set of the possible 2-,3-, and 4-mers is also synthesized. A 4-pattern mask was used. To makethe 6-mers in a 7-mer kernel, a 5-pattern mask is used. To make the7-mers in an 8-mer kernel, a 6-pattern mask is used, etc. To make allthe 7-mers in a 12-mer kernel, a 6-pattern mask is still all that isneeded. As a "bonus," all of the truncation sequences are generated withthis strategy of letting the masks extend beyond the "desired" regions.

For example, FIG. 2 illustrates that in a 6-monomer sequence the 1- to5-position substituted polymers are formed in the primary regions ofinterest (indicated by arrows), the 2- to 6-position substitutedpolymers are formed in region 403, the 3- to 6-position substitutedpolymers are formed in region 404, the 4- to 6-position substitutedpolymers in region 405, etc. The 1- to 4-position substituted polymersare formed in regions 406, the 1- to 3-position substituted polymers inregion 407, etc.

In some embodiments, the substrate is only as large as the regionindicated by arrows. It will often be desirable, however, to synthesizeall of the molecules illustrated in FIG. 2 since the deletion sequencesand others found outside of the region delineated by arrows will oftenprovide additional valuable binding information.

As shown, a single mask 401 is used for all of steps 1-5, while anothermask 402 is used for steps 6-11. The same mask 401 is used for steps12-16, but the mask is rotated preferably 180 degrees with respect tothe substrate. The light tranmissive regions of the mask 401 extend thefull length ("Y") of the area of interest in the y-direction. As shown,in step 1, the mask is placed above the substrate, the substrate isexposed, and the A monomer is coupled to the substrate in selectedportions of the substrate corresponding at least to the region 401a.Monomer A may also be placed at other locations on the substrate atpositions corresponding to mask regions 401b, 401c, and 401d.Alternatively, these regions of the mask may simply illuminate regionsthat are off of the substrate or otherwise not of interest. If theseregions correspond to regions of the substrate, various truncatedanalogs of the sequences will be formed.

Thereafter, as shown in step 2, the same mask is utilized, but it istranslated to the right. The substrate is exposed by the mask, and themonomer B is then coupled to the substrate. Thereafter, coupling steps3, 4, and 5 are conducted to couple monomers C, D and E, respectively.These steps also use the same mask translated to the right in the mannershown.

Thereafter, mask 402 is used for the "X" coupling steps. The mask 402contains a single stripe that extends the full length ("X") of the areaof interest in the x-direction. Mask 402 is preferrably a single, linearstripe that will normally be of width Y divided by the number ofmonomers in the substitution basis set. For example, in the case of 6amino acids as a basis set for the monosubstitution of peptides, thestripe will have a width of Y/6. The mask is repeatedly used to coupleeach of the monomers in the basis set of monomers and is translateddownwards (or upwards) after each coupling step. For example, the maskmay be placed at the top of the region of interest for the firstcoupling step, followed by translation downwards by 1/6 of the Ydimension for each successive coupling step when 6 monomers are to besubstituted into the target. FIG. 2 shows only 2 mask steps forsimplicity, but a greater number will normally be used.

Thereafter the mask 401 is again utilized for the remaining couplingsteps. As shown, the mask 401 is rotated, preferably 180 degrees, forthe following coupling steps. The succeeding coupling steps 12-16 areused to couple monomers B-F, respectively. The resulting substrate isshown in "cross section" in the bottom portion of FIG. 2. Again, theprimary area of interest is designated by arrows and may be the onlyregion used for synthesis on the substrate. The truncated substitutionsoutside of this region will also provide valuable information, however.

One extension of this method provides for the synthesis of all thepossible double substitutions of a kernel sequence. For a kernelsequence 7 residues long, there are 8400 peptides that make up allpossible disubstitutions of 20 amino acids, not considering replication(8380 unique). These peptides can be synthesized in 55 steps with 17masks. The synthesized sequences are shown below:

    __________________________________________________________________________    G G G G G X G G G G X G G G X G G X G X X                                     F F F F X F F F F X F F F X F F X F X    F                                                                        X                                         E E E X E E E E X E E E X E E X E E X    X                                                                        E                                         D D X D D D D X D D D X D D D X X X D    D                                                                        D                                         C X C C C C X C C C C X X X X C C C C    C                                                                        C                                         X B B B B B X X X X X B B B B B B B B    B                                                                        B                                         X X X X X X A A A A A A A A A A A A A    A                                                                        A                                         __________________________________________________________________________

The systematic substitution of three or more positions of the kernelsequence is also easily derived. The optimum polymer identified from theabove strategy can then serve as the new kernel sequence in furtheriterations of this process. The present method may be used for anydesired systematic substitution set, such as all 8-mers in a 12-merkernel, substitution in cyclic polymers, and the like. This methodprovides a powerful technique for the optimization of ligands that bindto a molecular recognition element.

B. Cyclic Polymer Mapping

Copending application Ser. No. 07/796,727 (Entitled "Polymer Reversal onSolid Surfaces"), incorporated herein by reference for all purposes,discloses a method for forming cyclic polymers on a solid surface.According to one aspect of the present invention, improved strategiesfor forming systematically varied cyclic polymers are provided.

In the discussion below, "P" refers to a protecting group, and X, Y, andZ refer to the various reactive sites on a tether molecule T. A, B, C,D, E, and F refer to various monomers or groups of monomers. Tosynthesize a cyclic polymer according to one aspect of the inventionherein, the process is conducted on a substrate. A tether molecule T iscoupled to a surface of the substrate. T may be one of the monomers inthe polymer, such as glutamic acid in the case of amino acids. Otherexamples of amino acid tether molecules include, but are not limited to,serine, threonine, cysteine, aspartic acid, glutamic acid, tyrosine,4-hydroxyproline, homocysteine, cysteinesulfinic acid, homoserine,ornithine, and the like. The tether molecule includes one or morereactive sites such as a reactive site Z which is used to couple thetether to the substrate. The tether also includes a reactive site Xhaving a protecting group P₂ thereon. The tether molecule furtherincludes a reactive site Y with a protecting group P₁ thereon.

In a first step, a polymer synthesis is carried out on the reactive siteY. According to some embodiments, conventional polymer synthesistechniques are utilized such as those described in Atherton et al.,previously incorporated herein by reference for all purposes. A widevariety of techniques may be used in alternative embodiments. Forexample, according to one embodiment, a variety of polymers withdifferent monomer sequences are synthesized on the substrate. Suchtechniques may involve the sequential addition of monomers or groups ofmonomers on the growing polymer chain, each monomer of which may alsohave a reactive site protected by a protecting group.

A variety of such methods are available for synthesizing differentpolymers on a surface. For example, Geysen et al., "Strategies forEpitope Analysis Using Peptide Synthesis," J. Imm. Meth., (1987)102:259-274, incorporated herein by reference for all purposes,describes one commonly used technique for synthesizing differentpeptides using a "pin" technique. Other techniques include those ofHoughten et al., Nature (1991) 354:84-86, incorporated herein byreference. In some embodiments, advanced techniques for synthesizingpolymer arrays are utilized, such as those described in copendingapplication Ser. No. 07/796,243, or light-directed,spatially-addressable techniques disclosed in Pirrung et al., U.S. Pat.No. 5,143,854; U.S. application Ser. No. 07/624,120; and Fodor et al.,"Light-Directed Spatially-Addressable Parallel Chemical Synthesis,"Science (1991) 251:767-773, all incorporated herein by reference for allpurposes, such techniques being referred to herein for purposes ofbrevity as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis)techniques.

During polymer synthesis, the activator used to remove P₁ (if any) onthe Y reactive site, and on reactive sites of the growing polymer chain,should be different than the activator used to remove the X protectinggroup P₂. Merely by way of example, the activator used to remove P₂ maybe a first chemical reagent, while the activator used to remove theprotecting group P₁, may be a second, different chemical reagent such asacid or base. By way of further example, the activator used to removeone of the protecting groups may be light, while the activator used toremove the other protecting group may be a chemical reagent, or bothactivators may be light, but of different wavelengths. Of course, othercombinations will be readily apparent to those of skill in the art onreview of this disclosure.

By virtue of proper protecting group selection and exposure to only theP₁ activator, the reactive site X is protected during polymer synthesisand does not take part in the initial portion of the process. Also, thereactive site Y remains bound to the monomer A. The synthesis step ofthe process, which will frequently include many substeps ofdeprotection/coupling, results in a polymer of a desired length, such asABCD . . . F. A polymer with 5 or more monomers is used by way ofexample, but fewer (or more) monomers will be utilized according to someembodiments.

In a next step of the process, the protecting group P₂ on the X reactivesite is removed. In addition, the reactive site on the last monomer F isrendered active, if necessary. The reactive site on the selected monomerwill then react with the reactive site X, forming a cyclic polymer. In apreferred embodiment for peptide synthesis, the protecting group P₂ isremoved with light.

Choice of the various protecting groups will generally be dictated bythe type of polymer which is to be synthesized and the desired synthesistechnique. Therefore, for example, oligonucleotides will often havedifferent protecting groups than will peptides, oligosaccharides, andthe like. In addition, conventional solid-phase synthesis techniqueswithout the use of photoremovable protecting groups will utilizedifferent protecting groups than VLSIPS™ light-directed synthesistechniques. Specific examples of protecting groups are discussed indetail below. Table 1 summarizes the various protecting groups usedaccording to most preferred embodiments of the invention.

                  TABLE 1                                                         ______________________________________                                        Preferred Protecing Group Selections                                                        P.sub.1 /    P.sub.2 /                                          Synthesis     Activator    Activator                                          ______________________________________                                        Standard      FMOC/        NVOC                                               Peptide       Base         or other                                                                      photochemical/                                                                base or light                                      Standard      BOC/         NVOC                                               Peptide       Acid         or other                                                                      photochemical/                                                                base or light                                      Standard      DMT/         NVOC                                               Nucleotide    Mild Acid    or other                                                                      photochemical/                                                                light                                              VLSIPS ™   NVOC (or     FMOC, allyl,                                       Peptide       other photo- silyl, or other                                                  chemical     base sens./                                                      protecting   base                                                             groups)/                                                                      Light                                                           VLSIPS ™   NV or        DMT                                                Nucleotide    NVOC/        or other                                                         Light        acid sens./                                                                   acid                                               ______________________________________                                    

One technique of standard Merrifield peptide synthesis employsfluorenylmethyloxycarbonyl (FMOC) on the growing end (amino terminus) ofthe polymer and one or more of a variety of side chain protectinggroups. According to preferred embodiments herein, such techniquesgenerally utilize mild base treatment to remove the FMOC (P₁) forpeptides and strong acid (up to 100% TFA) for both removal of the sidechain protecting groups and cleavage of the tether/polymer bond. Base orlight is used to remove the protecting group P₂, which may be, forexample, NVOC.

One embodiment of the invention utilizes a group P₁ which is removablewith a first wavelength of light and a second photoclearable group P₂which requires a different wavelength for deprotection of X. Preferablysuch groups utilize wavelengths >300 nm to avoid conflicting withprotecting groups in use during polymer synthesis and to avoid damage tosensitive amino acids. Alternatively, some embodiments employ a base-,paladium- or fluoride-sensitive protecting group. Other such materialsinclude FMOC, β cyanoethyl, t-butyldiphenylsilyl, allyl and othersapparent to those of skill in the art.

Cyclic polymers immobilized on a surface present unique opportunitiesfor biological activity screening. For example, with a cyclic polymer itis desirable to vary not only the monomers in the polymer, but also thepoint at which the cyclic polymer attaches to the substrate and,therefore, the region of the polymer available for binding. Further,depending on the building blocks of the polymer, one or more of thebuilding blocks may not be amenable to attachment to a substrate.

FIG. 3 illustrates this aspect of the invention. In the particularembodiment shown in FIG. 3, a cyclic polymer made from 8 monomers isillustrated. As shown therein, it is desirable to synthesize the polymerso that it is attached to the substrate at different positions in thering. For example, the left-most molecule in FIG. 3 is attached to thesubstrate with monomer 1, the second polymer is synthesized such that itis attached to the substrate via monomer 2, and the third polymer isattached to the substrate via monomer 3. In the most general case, it isdesirable to synthesize an array of cyclic polymers in which a polymeris attached to the substrate via each of the monomer positions. Forexample, with reference to FIG. 3, it is desirable to attach the8-monomer polymer via each of the 8 monomers therein. The site availablefor recognition will be slightly different for the polymer for eachattachment position, because the polymer is presented in a "rotated"position on the various regions of the substrate.

Again, while the invention is illustrated with regard to cyclic polymerswith about 8 monomers, a wide range of polymers may be utilized inconjunction with the invention without departing from the scope thereof.For example, when the polymers are peptides, the polymer molecules willtypically contain between about 4 and 10 monomers, often between about 6and 8 monomers.

Difficulties arise, however, in the attachment of certain monomers tothe substrate. For example, in the case of amino acids, certain aminoacids do not have side groups that are amenable to attachment to a solidsubstrate. Accordingly, in one embodiment of the invention, an array ofcyclic polymers is synthesized in which the monomer used for attachmentto the substrate is readily attachable to the substrate, such asglutamic acid. Like the first embodiment, the polymers have a commonmonomer sequence. However, the monomer used for coupling in all of thesites, according to this embodiment, is a common monomer that is easilycoupled to the substrate.

The substrate attachable monomer may either be substituted into thenative polymer sequence at various locations by alternatively deletingone member of the polymer chain or by inserting the substrate attachablemonomer into the native polymer sequence at different locations. FIGS. 4and 5 illustrate these alternatives, with FIG. 4 illustrating asubstitution strategy (in which a selected tether molecule issubstituted into the native ring at the coupling site), and FIG. 5illustrating an insertion strategy (in which a selected tether moleculeis added to the native ring at the coupling site).

As shown in FIG. 4, substrate attachable monomer "A," referred toelsewhere herein as a tether, is coupled to the substrate and links thecyclic polymer to the substrate. In preferred embodiments, A substitutesfor the monomer that would otherwise be in the position of the polymerthat is coupled to the substrate. This substitution preserves theoriginal ring size. For example, in the cyclic polymer molecule in theleft portion of FIG. 4, the monomer A has been substituted for themonomer 1. The monomer A has been substituted for the monomer 2 in thepolymer in the second portion of FIG. 4. The monomer A has beensubstituted for the monomer 3 in the polymer in the next polymer of FIG.4.

In most embodiments, it will be desirable to synthesize an array ofcyclic polymers in which the polymers are coupled to the substrate ateach monomer position in the polymer. For example, in the case of an8-mer, there will be 8 different attachment positions for the polymer.Often, it will be desirable to synthesize arrays of polymerssimultaneously in which not only is the attachment position varied foran individual monomer, but different polymer molecules are formed on thesubstrate. For example, in the case of a cyclic pentapeptide with allpossible combinations of natural amino acid monomers and all possibleattachment positions, it may be desirable to synthesize all 800,000combinations of sequence and attachment locations on one or moresubstrates.

According to a preferred aspect of the invention, a single mask may beused to form cyclic polymers with varying points of attachment on asubstrate. FIGS. 6A to 6E illustrate one preferred masking strategy,using a 7-monomer cyclic polymer as an example. Formation of the rotatedcyclic polymers on the same substrate with formation of polymers havingdifferent monomers at the various positions of the polymers represents apreferred embodiment of this invention. Only those steps relevant to theformation of rotated cyclic polymers are outlined below for the purposeof simplicity in the illustration.

As shown in FIG. 6A, the process begins with the illumination andcoupling of molecule A to the surface in a region of interest 281.Molecule A may be attached directly or indirectly (via linkers) to thesolid substrate. Molecule A is provided with terminal protecting groupP₁ and side protecting group P₂ if necessary. P₁ and P₂ are removableunder different conditions. For example, in a preferred embodiment ofthe invention P₁ is removable upon exposure to light and P₂ is removableupon exposure to, for example, acid or base. Details of various tethermolecules A and protecting groups are included in copending applicationSer. No. 07/796,727, previously incorporated herein by reference.

Thereafter, as shown in FIG. 6B, the mask is translated so as to exposeonly the left-most portion of the region of interest, and monomer 1 iscoupled to the substrate. Monomer 1 also has a photoprotecting group ona terminus thereof. As shown, monomer 1 may also be coupled on areas ofthe substrate that are not in the region of interest, or the region tothe left of the region of interest may be off of the edge of thesubstrate. The left and right portions of the substrate will,accordingly, be ignored in the illustrations below.

Thereafter, the process continues as illustrated in FIG. 6C, in whichmonomer 2 is coupled to the growing polymer chain using the same mask,again translated by one position. Processing continues with successiveexposures to light using the translated mask, followed by coupling ofthe appropriate monomers, resulting in the substrate shown in FIG. 6D.Thereafter, the photoprotecting groups are removed from the terminus ofall of the terminal monomers.

Then, the side chain protecting groups are removed from each of thetether molecules A, followed by coupling of the terminal monomers to theformerly protected side chain groups, in accordance with the teachingsof Ser. No. 07/796,727. Accordingly, an array of polymers is produced asshown in FIG. 6E that contains spatially addressable regions containingthe cyclic 7 member polymer, each coupled at a different position in thepolymer via a common tether molecule.

In some embodiments it may be desirable to identify a target cyclicpolymer, and synthesize an array of cyclic polymers in which not only isthe polymer rotated, but also in which the monomers are systematicallysubstituted with various monomers from a basis set. For example, it maybe desirable to synthesize all polymers in which the 6th building blockis systematically substituted with 20 L-amino acids.

In accordance with this aspect of the invention, an "X" mask is includedin the synthesis strategy, similar to the method described above forlinear polymers. The X mask is used to couple each of the monomers froma basis set in a selected position of the cyclic polymer. Accordingly,the resulting array in preferred embodiments contains all of thepolymers in which one position is systematically varied. In addition,for each polymer, the polymer is coupled via each position in themonomer to the substrate.

For purposes of illustration, a cyclic polymer of 7 residues is shown.FIGS. 7A to 7C illustrate one preferred masking strategy for formingsuch cyclic polymer arrays. The particular embodiment shown in FIG. 7illustrates substitution of monomers at the "6" position. As shown inFIG. 7A, processing of the substrate is initially the same as that shownin FIG. 6, in which the A tether as well as monomers 1-5 are coupled tothe substrate. As shown in FIG. 7B (top view), the substrate is thenprocessed with an "X" mask. The X mask is used to couple the variousmonomers from a basis set to the substrate. The X mask may be the samefor coupling of each member of the basis set and is simply translatedfor the coupling. For example, in the first exposure, the X mask isarranged in the form of a horizontal rectangle at the top of the regionof interest on the substrate, and the first monomer from the basis set(X₁) is coupled in this region. The X mask traverses each of the regionsshown in FIG. 7A. The mask is then translated downwards, and the secondmonomer in the basis set (X₂) is coupled in this region, again with themask traversing each of the regions formed up to the step shown in FIG.7A. The mask is translated successively downwards until, for example,all 20 monomers in the basis set are coupled at various regions of thesubstrate, as indicated in FIG. 7B.

Thereafter, the original mask is again utilized. The mask is first usedto couple monomer 7 to the right 5/6 of the substrate shown in FIG. 7B.The mask is then translated and used to couple monomer 1 to the right4/6 of the substrate shown in FIG. 7B. The mask is then used to couplemonomer 2 to the right 3/6 of the substrate shown in FIG. 7B. Successivecouplings are conducted with monomers 3, 4, and 5. The sequences ofmonomers on the resulting substrate are illustrated below, where Xindicates that a region of the substrate contains polymers with each ofthe basis set of monomers selected for insertion at the 6th position ofthe polymer. The polymer sequences are obtained by examination of thecolumns of the illustration below:

    ______________________________________                                        X      7          1     2         3   4                                       5      X          7     1         2   3                                       4      5          X     7         1   2                                       3      4          5     X         7   1                                       2      3          4     5         X   7                                       1      2          3     4         5   X                                       A      A          A     A         A   A                                       ______________________________________                                    

Thereafter, the polymers are cyclized, resulting in an array of polymersin which every member of the basis set is inserted at the sixth positionof the polymers, and in which each polymer thus synthesized is coupledto the substrate at each rotational position in the polymer, all atspatially addressable regions. A portion of the resulting array isillustrated in FIG. 7C.

To vary every building block at every position systematically requires adifferent set of masks. For the above 7-numbered cyclic peptide, thelength of the synthesis region is divided into 36 equal units, while thewidth of the synthesis region is divided into "x" units (typically x=20for peptides with "natural" amino acids). The resulting polymer librarywill be as follows, again with columns indicating the resulting polymersequence and "X" indicating that polymers with all members of a basisset substituted at that position are formed:

    ______________________________________                                        66666X11111X22222X33333X44444X55555X                                          5555X56666X61111X12222X23333X34444X4                                          444X44555X55666X66111X11222X22333X33                                          33X33344X44455X55566X66611X11122X222                                          2X22223X33334X44445X55556X66661X1111                                          X11111X22222X33333X44444X55555X66666                                          AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA                                          ______________________________________                                    

In synthesizing the above polymers, the order of coupling will be:A1234561234X2345612345. FIG. 8 illustrates the cyclic polymers resultingfrom the synthesis. FIG. 9 illustrates a convenient mask used in thesynthesis.

This strategy differs slightly from those discussed above in that thesynthesis produces 7-membered rings in which the tether polymer is addedto 6-membered kernel sequence. To mimic the structure of the actualpolymer more closely, it is desirable in some embodiments to use atether molecule that is shorter than the monomer molecules so as tomaintain the native length of the molecule. For example, in the case ofpeptides, a disulfide molecule serving as the tether will be beneficial.Examples of such cyclic peptides with a disulfide linkage are common inthe literature, i.e., oxytocin and vasopressin. The 6 amino acids andthe disulfide linkage will produce a 20-membered ring. By comparison, acyclic hexamer of a peptide is an 18-membered ring, while a cyclicpeptide heptamer will be a 21-membered ring. Accordingly, when thedisulfide molecule is inserted into the cyclic polymer, the tether willproduce polymers that more correctly mimic the native polymer. Otheruseful tether molecules include glutamic acid, as illustrated in FIGS.10A and 10B.

The following series of polymers can be made with the same masks (one tolay down "A", two with 180° symmetry to assemble the polymer, and one"X" mask). This method produces all single substitutions possible whilemaintaining the rest of the cycle constant. One could also use themethod to make double and triple substitutions.

    __________________________________________________________________________    77777X11111X22222X33333X44444X55555X66666X                                    6666X67777X71111X12222X23333X34444X45555X5                                    555X55666X66777X77111X11222X22333X33444X44                                    44X44455X55566X66677X77711X11122X22233X333                                    3X33334X44445X55556X66667X77771X11112X2222                                    X22222X33333X44444X55555X66666X77777X11111                                    AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA                                    __________________________________________________________________________

In the above set of polymers A is the molecule used to couple thepolymer to the substrate, 1-7 are amino acids or other monomers in thekernel sequence, and X are the substitutions from the basis set ofmonomers. One way to assemble this array is: A23456712345X34567123456.The masks that would be utilized before the X coupling are shown in FIG.11A, and the masks used after the X coupling are shown in FIG. 11B.

III. Data Collection

A. CCD Data Collection System

Although confocal detection systems are typically used for datacollection, according to one embodiment a high resolution CCD camerasystem is utilized for data collection. The camera allows for thedigitization of images with a resolution of, e.g., 1300 by 1024 pixels.A dynamic range of >60 dB can be obtained if the sensor is calibratedwith respect to dark current and gain. Cooling the camera to 248° Klowers the dark current to a tolerable level even when prolongedexposure times (several minutes) are needed.

According to one embodiment, a 100 W Hg-Arc lamp is used as a lightsource. The infrared components of the light are blocked using aheat-absorbing filter. A second filter is used to select the excitationwavelength using an optional ground glass plate filter. Best results areachieved by illuminating the sample with UV. Although the optimumexcitation wavelength for FITC lies at 490 nm, excitation in the UVrange shows better results than with a 490 nm IF filter, because the Hglamp provides more optical energy in the UV band. The sample isilluminated at an angle of 45° with respect to the light beam and theCCD camera. This ensures that no direct light path exists between thelamp and the sensor and therefore reduces background radiation.

The CCD camera is mounted at the back of a Hasselblad 500 C/M camerasystem with a lens and IR filter. An automatic bellow and a standard 80mm (f 2.8) lens are used. This system results in an imaging scale whichis selectable between 1:3 and 1:0.8. The sample plus the filters arehoused in a case to prevent light from the surroundings from enteringthe optical path. Digitized image data are transmitted to a 386 PC,where the images can be viewed on a high resolution display.

Longer integration times (up to 2 minutes) yield better S/N ratios. Oneproblem is the fluorescence (and light diffraction) of dust particles onthe surface of the chip used for the Very Large Scale ImmobilizedPolymer Synthesis (VLSIPS™) techniques. Dust particles deliver a signalabout 10 times higher than the FITC fluorescence. Therefore, theintegration time can be increased only as long as the dust particles donot cause an overflow (blooming) of the sensor. However, since an areaof about 50×50 pixels can be averaged in the digitized image for aquantitative assessment of the fluorescence, the measurement accuracy issufficient with integration times between 15 and 30 seconds.

If the acquisition time needs to be lower than the 15-20 secondsmentioned above, a different light source should be selected. An argon(λ=488 nm) laser is optimal. The disadvantage of using a laser is theconsiderably higher technological expense, and the possibility ofbleaching if the optical energy is raised too much. Bleaching could notbe observed while using the Hg lamp (even with integration times ofseveral minutes). Using a lens with a lower focal length (e.g., astandard 50 mm/f 1.4 lens) would improve the overall efficiency of thesystem.

The optimized optical system could allow for measurement times below 10seconds using a Hg lamp. When using a laser, image acquisition will takeless than 1 second. The system should be operated in a dust-freeenvironment (such as a lateral flow workbench) to reduce errorsgenerated by dust particles.

B. Trapping Low Affinity Interactions

According to one embodiment, the invention provides a methodology forchemically trapping low affinity interactions between receptors andimmobilized ligands. Monovalent receptors with K_(d) 's greater than 100nM may not bind with sufficient affinity to an immobilized ligand tosurvive subsequent washing and imaging steps for later detection. Thus,while high concentrations (approximately near the K_(d) of the peptidelead) of receptor should bind to some epitopes on, for example, aVLSIPS™ chip, this information may be lost during subsequent processing.Accordingly, cross-linkers are used according to one embodiment of theinvention. The cross-linkers are designed to be specific for thereceptor-ligand complex while having relatively no specificity for freereceptor. Accordingly, it is possible not only to trap covalently thereceptor, but also recover the excess receptor in unmodified form.

To accomplish this the invention provides:

1. A residue or "handle" common to all ligands on the solid support.

2. Heterobifunctional crosslinking agents in which one of thefunctionalities alkylates the "handle" kinetically much faster than itwould alkylate either the receptor or the immobilized ligand.

3. The second functionality of the heterobifunctional crosslinkeralkylating the immobilized receptor kinetically much faster than itwould alkylate either the free receptor or the immobilized ligand.

According to one embodiment, the substrate is "doped" by replacing asmall amount of the NVOC-aminocaproic acid reagent on the surface of thesubstrate with a small quantity of t-Boc-mercaptocaproic acid. Themercaptan would be blocked during all of subsequent peptide synthesissteps, but would deprotect upon exposure to acid. The surface would thenbe treated with receptor solution and the free sulfhydryl group acts asan outstanding nucleophile that is alkylated instantaneously upontreatment with a variety of commercially available heterobifunctionalcrosslinkers. In a second crosslinking step, alkylation of boundreceptor is facile, because the proximity of the protein makes thisreaction pseudo first-order. Nonspecific alkylation of free receptorwould be a second-order process and therefore disfavored over specificalkylation. The excess receptor would then be removed and recovered byfast-desalting chromatography.

Because the receptor is now covalently bound to the substrate,subsequent processsing will not remove the receptor enabling detectionof event those receptors with a high Kd.

C. Fluorescence Energy-Transfer Substrate Assays

A different application of the present invention tests for catalyticcleavage of various polymer sequences by an enzyme or other catalyst.For example, aspartyl proteases such as renin, HIV proteases, elastase,collagenase and some cathepsins can be tested against an array ofpeptides. According to this aspect of the invention, a variety ofpeptide sequences are synthesized on a solid substrate by theprotection-deprotection strategy outlined above. The resulting array isprobed with an enzyme which might cleave one or more peptide elements ofthe array resulting in a detectable chain.

In one embodiment, the peptides to be tested have a fluorescence donorgroup such as 1-aminobenzoic acid (anthranilic acid or ABZ) oraminomethylcoumarin (AMC) located at one position on the peptide and afluorescence quencher group such as lucifer yellow, methyl red ornitrobenzo-2-oxo-1,3-diazole (NBD) at a different position near thedistal end of the peptide. Note, that some "donor" groups can also serveas "quencher" groups, depending on the relative excitation and emissionfrequencies of the particular pair selected. The intramolecularresonance energy transfer from the fluorescence donor molecule to thequencher will quench the fluorescence of the donor molecule. Uponcleavage, however, the quencher is separated from the donor group,leaving behind a fluorescent fragment. A scan of the surface with anepifluorescence microscope, for example, will show bright regions wherethe peptide has been cleaved. FIG. 12A shows a tripepride having adonor-quencher pair on a substrate. The fluorescence donor molecule,1-aminobenzoic acid (ABZ), is coupled to the ε-amino group of lysine(Lys) on the P' side of the substrate. The donor molecule could, ofcourse, be attached to the α-amine group. A fluorescence quencher, NBDcaproic acid, is coupled to the P side of the substrate molecule. Uponcleavage by a protease, as shown in FIG. 12B, the quencher is releasedleaving the fluorescent fragment still bound to the solid substrate fordetection.

FIGS. 13A to 13H illustrate various alternative donor/quencher pairswhich are used according to alternative embodiments of the invention.

IV. Examples

A. Example

The binding interactions of a monoclonal antibody D32.39, which wasraised against the opioid peptide dynorphin B (YGGFLRRQFKVVT) (SEQ IDNO: 3), were explored. The binding of antibody D32.39 to dynorphin B waspreviously shown to be directed to the carboxyl terminus. Initially, theexperiment addressed the minimum peptide size required for binding andthe location of the antibody binding epitope within the full lengthpeptide. A binary masking strategy was used to generate an array ofpeptides of all the linear sequences contained within the terminal tenresidues (FLRRQFKVVT). The ten-step synthesis was configured to yieldfour replicates of the 1024 possible compounds (i.e., 4096 distinctpolymer synthesis regions), of which 1023 were peptides ranging inlength from one to ten residues in length, with a mean length of five.This array includes every possible truncation and deletion sequence, aswell as multiple deletions, contained within these ten residues, whilepreserving the linear order of amino acids. Due to the redundancy inamino acids (two valines, two arginines, and two phenylalanines), 560unique sequences were generated.

After synthesis of the array, the terminal NVOC protecting groups wereremoved, the terminal amines were acetylated, and the side chainprotecting groups were cleaned under standard conditions. Non-specificprotein binding to the surface was blocked with 1% BSA/PBS/0.05% TWEEN20™ (polyoxyethylenesorbitan monolaurate). The array was incubated withthe D32.39 antibody at a concentration of 10 μg/ml for 2 hours, followedby reaction with 10 μg/ml of FITC conjugated anti-mounse antibody(Sigma) for 2 hours at 20° C. Following extensive washing with buffer,the array was scanned in a confocal fluorescence microscope with 488 nmexcitation from an argon ion laser (Spectra-Physics). The resultantfluorescence image containing the intensity versus positional data forthe array of peptides was normalized from 0 to 100% relative intensityby subtracting out background (the region within the array containingonly the linker molecule) and using the region of highest intensity(FRQFKVVT) (SEQ ID NO: 4) as 100% relative intensity. The replicateswere averaged, and the data sorted according to desired sequences. Theentire screening process, including peptide synthesis and dataacquisition and workup, required three days to complete.

A survey of the data obtained from the ten possible single deletionpeptides affords a preliminary estimate of the regions within the kernelsequence responsible for binding to the antibody. The normalizedfluorescence intensity for each of these deletion sequences are is inFIG. 14. The full length peptide exhibited about 100% relative signal asanticipated, indicating that the epitope lay within the 10-mer sequence.Deletions in the kernel sequence near the amino terminus had littleeffect on the observed signal (compare F, L, R, R deletions), whiledeletions near the carboxyl terminus (compare F, K, T deletions) had amore pronounced effect. The antibody shows intermediate relative bindingto both the valine and glutamine deletions.

An analysis of the terminal truncated peptides generated in the arrayallows one to draw conclusions regarding the size and location of theepitope contained in the kernel sequence. The normalized fluorescencesignals obtained for the terminal truncated peptides are shown in FIG.15. The data reveals that truncations from the amino terminus aretolerated until loss of the second arginine residue, indicating theimportance of the arginine residue to antibody recognition. Truncationsfrom the carboxyl terminus are not tolerated as well, and the initialtruncation of the threonine residue results in a large decrease in theobserved fluorescence signal. The combination of these two observationspredicts that the epitope lies between, and includes both, the arginineand threonine residues, or hence RQFKVVT (SEQ ID NO: 5).

Examination of the signals observed for all the possible truncatedsequences in Table 2 contained within the kernel sequence affords thehighest degree of confidence in assigning the size and position of theepitope. Peptides shorter than seven residues were observed to showdiminished binding to the antibody. Binding of the D32.39 antibody tothe immobilized peptides exhibits a strong bias towards the RQFKVVTsequence.

                  TABLE 2                                                         ______________________________________                                        Sequence and Relative Fluorescence Intensities                                of the Truncated Dynorphin Peptides                                           Sequence          Normalized Intensity (%).sup.a                              ______________________________________                                        FLRRQFKVVT        98 ± 1                                                    LRRQFKVVT (SEQ ID NO: 6)                                                                        87 ± 13                                                 FLRRQFKVV (SEQ ID NO: 7)                                                                         37 ± 15                                                   RRQFKVVT (SEQ ID NO: 8)                                                                       99 ± 1                                                    LRRQFKVV (SEQ ID NO: 9)                                                                         44 ± 12                                                 FLRRQFKV (SEQ ID NO: 10)                                                                        14 ± 3                                                      RQFKVVT (SEQ ID NO: 11)                                                                      99 ± 1                                                     RRQFKVV (SEQ ID NO: 12)                                                                        58 ± 10                                                  LRRQFKV (SEQ ID NO: 13)                                                                        16 ± 3                                                   FLRRQFK (SEQ ID NO: 14)                                                                         10 ± 3                                                       QFKVVT (SEQ ID NO: 15)                                                                      28 ± 3                                                      RQFKVV (SEQ ID NO: 16)                                                                        54 ± 11                                                   RRQFKV (SEQ ID NO: 17)                                                                        16 ± 3                                                    LRRQFK (SEQ ID NO: 18)                                                                         12 ± 3                                                   FLRRQF (SEQ ID NO: 19)                                                                           8 ± 3                                                        FKVVT (SEQ ID NO: 20)                                                                      13 ± 4                                                        QFKVV (SEQ ID NO: 21)                                                                      17 ± 2                                                      RQFKV (SEQ ID NO: 22)                                                                        16 ± 4                                                     RRQFK (SEQ ID NO: 23)                                                                         12 ± 3                                                    LRRQF (SEQ ID NO: 24)                                                                          10 ± 4                                                   FLRRQ (SEQ ID NO: 25)                                                                            8 ± 3                                                         KVVT (SEQ ID NO: 26)                                                                      11 ± 2                                                        FKVV (SEQ ID NO: 27)                                                                       10 ± 2                                                       QFKV (SEQ ID NO: 28)                                                                        11 ± 2                                                      RQFK (SEQ ID NO: 29)                                                                         13 ± 5                                                     RRQF (SEQ ID NO: 30)                                                                          11 ± 5                                                    LRRQ (SEQ ID NO: 31)                                                                           10 ± 4                                                   FLRR (SEQ ID NO: 32)                                                                             6 ± 3                                                   ______________________________________                                         .sup.a Errors were standard deviations of the averaged signals from a         minimum of four replicates. All peptides were acetylated on the amino         terminus and were linked to the surface via an amide bond to the carboxyl     terminus.                                                                

To confirm the interpretation of the observed fluorescence signals,peptides synthesized on a conventional solid phase peptide synthesizerwere tested. The IC₅₀ values for the competition of free peptide againstradiolabeled dynorphin B peptide were determined and are tabulated inTable 3. There is a striking correlation between the rank ordering ofthe relative fluorescence intensity and the solution IC₅₀ values.Although the antibody appears to require the presence of the threonineresidue at the carboxyl terminus, it shows little preference for thefree carboxamide versus the free acid.

                  TABLE 3                                                         ______________________________________                                        Solution Binding Data for the                                                 Dynoriphin Peptides to the D32.39 Antibody                                                              Normalized Intensity                                Sequence        IC.sub.50 (μM).sup.a                                                                 (%).sup.b                                           ______________________________________                                        YGGFLRRQFKVVT-OH.sup.                                                                         0.0057    nd.sup.c                                            Ac-FLRRQFKVVT-OH.sup.                                                                         nd        98                                                  Ac-RRQFKVVT-OH.sup.                                                                           0.0039    99                                                  Ac-RQFKVVT-OH.sup.                                                                            0.011     99                                                  Ac-RQFKVVT-NH.sub.2                                                                           0.0073    ---                                                 Ac-QFKVVT-OH.sup.                                                                             3.2       28                                                  Ac-FKVVT-OH.sup.                                                                              77.0      13                                                  ______________________________________                                         .sup.a The IC.sub.50 values were determined by competition against            radiolabeled dynorphin B peptide.                                             .sup.b The normalized intensity refers to the relative fluorescence           intensity observed from the corresponding surfaceimmobilized peptides. Al     surfaceimmobilized peptides were acetylated on the amino terminus and wer     linked to the surface via an amide bond to the carboxyl terminus.             .sup.c Not determined.                                                   

The relative fluorescence intensity observed for biological recognitionof an antibody to an array of immobilized peptides depends on severalfactors. Of primary importance is the multivalent interaction betweenthe antibody and the surface due to the presence of two antibodycombining sites in an IgG molecule. If the peptide chains are spacedrelatively close on a surface, then the antibody can span two chains andthe observed effective binding constant may be greater than themonovalent value. Estimates of the surface density of the reactivepeptide chains on the surface suggest that this is likely to occur. Inaddition, the situation here is even more complex because a secondbivalent antibody was used to detect the initial binding of D32.39.

With the size and position of the epitope thus determined, the presentinvention was used to examine novel substitutions in the RQFKVVT peptidesequence. The methods of the invention allowed systematic replacement ateach position in the lead sequence by other amino acids. A schematic ofthe substitutions is shown in Table 4, where X represents the positionundergoing substitution. When X is the 20 L-amino acids, the arraycomprised 140 peptides which were synthesized and screened for binding.The masking technique illustrated in FIG. 1A-1C with a translated,full-size mask, as shown in step 2 of FIG. 1A, was utilized. Preliminaryresults identified Q, K, V, and T as the residues most amenable tosubstitution.

                  TABLE 4                                                         ______________________________________                                        Schematic of Illustrative Single Substitutions                                into the ROFKVVT Peptide Sequence                                             ______________________________________                                                    XQFKVVT.sup.a                                                                 RXFKVVT                                                                       RQXKVVT                                                                       RQFXVVT                                                                       RQFKXVT                                                                       RQFKVXT                                                                       RQFKVVX                                                           ______________________________________                                         .sup.a X represents the position undergoing substitution.                

These results demonstrate the application of a novel technique employingboth photolithography and solid phase peptide chemistry to create arraysof spatially-addressable chemical libraries. The ability to screensimultaneously all the immobilized peptides for binding to a biologicaltarget allows one to generate powerful structure activity relationship(SAR) databases. The use of novel building blocks as a tool to impartdesirable physical properties into the arrays should aid in theoptimization of new lead compounds in the area of drug discovery.

B. Example

The example below illustrates aspects of one methodology for theformation of cyclic polymers. The method may be used to construct arraysof cyclic polymers according to the above methods.

Eight slides derivatized with NVOC-aminocaproic acid werephotodeprotected for ten minutes in 5 mM sulfuric acid/dioxane using 365nm light. After neutralization of the surface, six of the slides wereexposed to 0.1M BOP activated NVOC-Glu(O-t-butyl)-OH, while theremaining two slides were exposed to 0.1M BOP activated NVOC-Glu-OFm.The first six slides were divided into three groups and each group wasderivatized with either BOP activated Boc-Pro-Pro-Pro-Pro-OH, (SEQ IDNO: 33), Boc-Ala-Ala-Ala-Ala-OH (SEQ ID NO: 34), orBoc-Ala-Gly-Gly-Gly-OH (SEQ ID NO: 35). The second two slides from abovewere derivatized with BOP activated Boc-Val-Val-Val-Val-OH (SEQ ID NO:36). This gave four pairs of slides, each with a pentapeptide on thesurface with a side chain carboxyl (still protected) with which tocyclize. Each slide was deprotected with TFA to remove the Boc andt-butyl groups (from both the amino terminus and the masked carboxylgroup), and then the two slides with Fm as a protecting group weretreated with piperidine to unmask the carboxyl group.

A sixteen-well template was placed on each slide in order to physicallysegregate different regions of the surface and one member of each pairwas warmed (either to 41° or 44° C.) while the second member was kept at20° C. during the following reactions. Each well of the template wastreated with either a 0.1M solution of activator or solvent for 4.5hours. The activators were BOP, HBTU, and diphenylphosphoryl azide(DPPA). After the specified time, the wells were washed and thetemplates removed. The slides were stained with a 10 mM solution of a9:1 mixture of phenyl isothiocyanate (PITC): fluorescein isothiocyanate(FITC). The slides were washed and scanned for fluorescence using aconfocal microscope.

Cyclization of the peptides was expected to result in the loss ofreactivity of the terminal amine, and hence, the loss of fluorescenceintensity. Cyclization efficiency was measured as the decrease influorescence intensity for the peptides that had been treated with anactivator as compared to untreated peptides. Cyclization was found tooccur readily in all cases. The activators BOP and HBTU were found to bemore effective than DPPA. Temperature had little effect on thecyclization efficiency.

V. Conclusion

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example., whilethe invention is illustrated primarily with regard to peptide,oligosaccharide and nucleotide synthesis, the invention is not solimited. By way of another example, while the detection apparatus hasbeen illustrated primarily herein with regard to the detection of markedreceptors, the invention will find application in other areas. Forexample, the detection apparatus disclosed herein could be used in thefields of catalysis, DNA or protein gel scanning, and the like. Thescope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 36                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       PheLeuArgArgGlnPheLysValValThr                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       PheLeuArgGlnPheLysValValThr                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TyrGlyGlyPheLeuArgArgGlnPheLysValValThr                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       PheArgGlnPheLysValValThr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgGlnPheLysValValThr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuArgArgGlnPheLysValValThr                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       PheLeuArgArgGlnPheLysValVal                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ArgArgGlnPheLysValValThr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       LeuArgArgGlnPheLysValVal                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      PheLeuArgArgGlnPheLysVal                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ArgGlnPheLysValValThr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ArgArgGlnPheLysValVal                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      LeuArgArgGlnPheLysVal                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      PheLeuArgArgGlnPheLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlnPheLysValValThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ArgGlnPheLysValVal                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ArgArgGlnPheLysVal                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      LeuArgArgGlnPheLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                     PheLeuArgArgGlnPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      PheLysValValThr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      Gl nPheLysValVal                                                              15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ArgGlnPheLys Val                                                              15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ArgArgGlnPheLys                                                               1 5                                                                           (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      LeuArgArgGlnPhe                                                               1 5                                                                           (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      PheLeuArgArgGln                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      LysValValThr                                                                  (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      PheLysValVal                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GlnPheLysVal                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ArgGlnPheLys                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       ArgArgGlnPhe                                                                 1                                                                             (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      LeuArgArgGln                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      PheLeuArgArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 4 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ProProProPro                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      AlaAlaAlaAla                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AlaGlyGlyGly                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       ValValValVal                                                                 1                                                                             __________________________________________________________________________

What is claimed is:
 1. A method of synthesizing an array ofoligonucleotides on a surface of a substrate, the method comprising thesteps of:identifying a target oligonucleotide having a nucleic acidmonomer sequence complementary to a receptor oligonucleotide ofinterest; synthesizing an array of oligonucleotides on said surface ofsaid substrate by the steps of activating predefined regions of saidsubstrate and covalently coupling selected nucleic acid monomers to saidsubstrate to form an array of oligonucleotides, said array ofoligonucleotides comprising more than one group of oligonucleotides,wherein each of said more than one group consists of a sequence ofnucleic acid monomers that is the same as the nucleic acid monomersequence of said target oligonucleotide, except that each of said morethan one group has a different single position in said nucleic acidmonomer sequence of said target oligonucleotide substituted with eachmember of a basis set of nucleic acid monomers; and determining whicholigonucleotide sequence in each of said groups of oligonucleotidesspecifically binds to said receptor oligonucleotide of interest.
 2. Themethod as recited in claim 1 wherein said determining step comprises thesteps of:contacting said array with said receptor oligonucleotide ofinterest; determining which oligonucleotide sequence in each of saidgroups of oligonucleotides in said array are complementary to saidreceptor oligonucleotide of interest by identifying where said receptoroligonucleotide of interest has bound said array.
 3. The method asrecited in claim 1 wherein said synthesizing step comprises synthesizingsaid more than one group of oligonucleotides by the steps of:performinga first series of activation and coupling steps in which each activationand coupling step in said first series of activation and coupling stepsactivates and couples nucleic acid monomers in said nucleic acid monomersequence of said target oligonucleotide in a selected region of saidsubstrate, each selected region comprising a portion of a regionactivated in a previous step of said first series of activation andcoupling steps; and performing a second series of activation andcoupling steps to couple each member of the basis set of nucleic acidmonomers to a different portion of the oligonucleotides formed in eachstep of said first series of activation and coupling steps.
 4. Themethod as recited in claim 1 wherein said activating steps comprise thestep of irradiating said substrate.
 5. The method as recited in claim 3wherein said activation steps comprise the step of irradiating saidsubstrate.
 6. The method as recited in claim 3, wherein saidsynthesizing step, further comprises the steps of performing a thirdseries of activation and coupling steps in which each of said activationand coupling steps couples nucleic acid monomers in said nucleic acidmonomer sequence of said target oligonucleotide to said oligonucleotidessynthesized in said first and second series of activation and couplingsteps.
 7. The method as recited in claim 5 wherein the substrate isplanar, and:said first series of irradiating steps uses a first maskhaving at least first and second transparent regions spanning a verticalportion of said substrate; and said second series of irradiating stepsuses a mask having a horizontally oriented transparent region spanningportions of said substrate corresponding to said first and secondtransparent regions.